Antitrypanosomal Activity of 1,2,3-Triazole-Based Hybrids Evaluated Using In Vitro Preclinical Translational Models

Simple Summary Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease that affects 6–7 million people worldwide. It is a global disease, due to migration from Latin America to other regions of the world, and a recognized worldwide public health problem. Clinical treatment is based on two fifty-year-old drugs, nifurtimox and benznidazole. These drugs have low efficacy in the chronic phase of the disease and have severe adverse effects, making the search for new drugs essential. This study aimed to evaluate the trypanocidal potential of 1,2,3-triazole analogs. Our data highlight three analogs with potent activity against trypomastigotes and similar efficacy to benznidazole, the reference drug, against intracellular parasites. These analogs showed high efficacy in 3D cardiac microtissue. However, despite potentially reducing parasite load, the promising candidates did not inhibit the resurgence of the parasite in the absence of the drug. Newly designed analogs will be screened against T. cruzi to identify potentially active and safe drugs for Chagas disease therapy. Abstract Chagas disease therapy still relies on two nitroderivatives, nifurtimox and benznidazole (Bz), which have important limitations and serious adverse effects. New therapeutic alternatives for this silent disease, which has become a worldwide public health problem, are essential for its control and elimination. In this study, 1,2,3-triazole analogues were evaluated for efficacy against T. cruzi. Three triazole derivatives, 1d (0.21 µM), 1f (1.23 µM), and 1g (2.28 µM), showed potent activity against trypomastigotes, reaching IC50 values 10 to 100 times greater than Bz (22.79 µM). Promising candidates are active against intracellular amastigotes (IC50 ≤ 6.20 µM). Treatment of 3D cardiac spheroids, a translational in vitro model, significantly reduced parasite load, indicating good drug diffusion and efficacy. Oral bioavailability was predicted for triazole derivatives. Although infection was significantly reduced without drug pressure in a washout assay, the triazole derivatives did not inhibit parasite resurgence. An isobologram analysis revealed an additive interaction when 1,2,3-triazole analogs and Bz were combined in vitro. These data indicate a strengthened potential of the triazole scaffold and encourage optimization based on an analysis of the structure–activity relationship aimed at identifying new compounds potentially active against T. cruzi.


In Silico Prediction
DataWarrior software version 5.5.0 [33] was used to predict physicochemical properties. SwissADME service (https:www.swissadme.ch accessed on 31 may 2023) was used to predict drug oral bioavailability using radar graphics.

Two-and Three-Dimensional Cell Cultures
VERO cells (Rio de Janeiro Cell Bank code 0245) were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2. Confluent monolayers were dissociated with trypsin-EDTA solution (0.025%), and isolated cells were seeded on culture plates or flasks, depending on the experimental assay. The VERO cell cultures were used in cytotoxicity and phenotypic screening assays and for obtaining culture-derived trypomastigotes.

In Silico Prediction
DataWarrior software version 5.5.0 [33] was used to predict physicochemical properties. SwissADME service (https:www.swissadme.ch accessed on 31 may 2023) was used to predict drug oral bioavailability using radar graphics.

Two-and Three-Dimensional Cell Cultures
VERO cells (Rio de Janeiro Cell Bank code 0245) were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 • C in a humidified atmosphere of 5% CO 2 . Confluent monolayers were dissociated with trypsin-EDTA solution (0.025%), and isolated cells were seeded on culture plates or flasks, depending on the experimental assay. The VERO cell cultures were used in cytotoxicity and phenotypic screening assays and for obtaining culture-derived trypomastigotes.
Primary heart muscle cell cultures were obtained from fetuses of female Swiss Webster mice, as previously described [34]. After heart removal and ventricle fragmentation, tissue fragments were dissociated in a trypsin and collagenase type II (Worthington Biochemical Corporation, Lakewood, USA) dissociation solution. For two-dimensional (2D) cultures, the isolated cardiac cells were seeded at a density of 5 × 10 4 cells/well in gelatin (0.1%)-coated 96-well white culture plates. Three-dimensional (3D) cardiac spheroids were obtained after seeding isolated heart muscle cells at a density of 2.5 × 10 4 cells/well in an agarose (1%)-coated 96-well U-bottom plate, as described in [35]. Heart muscle cells cultures were cultivated in Dulbecco's modified Eagle medium (DMEM) supplemented with 7% FBS, 2.5 mM CaCl 2 , 2% embryo extract, and 1 mM L-glutamin and maintained at 37 • C in a humidified atmosphere of 5% CO 2 . All procedures with animals were approved by the Animal Care and Use Committee at the Oswaldo Cruz Institute (license L-017/2022).

Parasites
A drug screen was carried out with Trypanosoma cruzi clone Dm28c genetically modified to express luciferase (Dm28c-luc), kindly provided by Dr. Cristina Henriques [36]. The genetically modified T. cruzi clone Dm28c (Dm28c-Luc) has the firefly luciferase gene integrated into the genome, stably expressing the luminescent enzyme. The bioluminescent signal, proportional to the number of parasites, is produced by adding a D-luciferin substrate [37]. Trypomastigotes were harvested from supernatants of VERO cell cultures, infected at a ratio of 10:1 parasite/host cells, 4 days post-infection (4 dpi). Parasites were used for phenotypic drug screening procedures. Luminescent parasites are a reliable and sensitive tool that allows precise quantification of parasite load. The use of genetically modified organisms was approved under license CQB 105/99.

Cytotoxicity Analysis
The toxic effect of the triazole series was evaluated on VERO cell monolayers. After seeding for 24 hours, VERO cells were treated for 72 h at 37 • C with the 1,2,3-triazole derivatives and Bz, the reference drug, with a range of concentration from 15.62 to 500 µM. Controls were performed with a non-toxic concentration of dimethyl sulfoxide (DMSO; ≤0.1%) used in the experimental assays. Cell viability was determined by measuring ATP level using a CellTiter Glo kit [38]. The luminescence read was performed using a Glomax microplate reader (Promega Corporation, Madison, WI, EUA). The cytotoxicity concentration 50 (CC 50 ), the drug concentration that reduces cell viability by 50%, was calculated using linear regression. The cardiotoxic effect of the most effective compounds was also evaluated using 2D and 3D cultures. Cultures were exposed for 72 h to compound concentrations up to 500 µM, following the protocol mentioned above. At least 3 independent assays were performed in duplicate.

Trypanocidal Activity
All triazole analogs were analyzed for their biological activity against trypomastigote and intracellular amastigote forms of T. cruzi (Dm28c-Luc). Trypomastigotes (1.0 × 10 6 parasites/well) were incubated for 24 h at 37 • C with triazole derivatives and Bz at concentrations ranging from 0.04 to 100 µM. Luciferin (300 µg/mL), a luciferase substrate, was added to parasite suspensions to evaluate trypomastigotes viability [38]. The luminescent signal was measured using a Glomax microplate reader. A maximal DMSO concentration (0.1%) was used as the negative control. The inhibitory concentration 50 (IC 50 ) and 90 (IC 90 ) against T. cruzi, which reduces the number of parasites by 50% and 90%, respectively, was calculated using linear regression. The selectivity index (SI), a ratio that measures the window between toxicity in mammalian cells and anti-T. cruzi activity (SI = CC 50 /IC 50 ), was also determined.
Activity against intracellular amastigotes was determined in cultures of VERO cells infected by T. cruzi (24 h), as previously described [38]. Briefly, infected cultures were treated for 72 h at 37 • C with different concentrations of triazole derivatives (0.04-100 µM). Then, the culture supernatant was removed, and the viability of the intracellular parasites was evaluated after adding luciferin (300 µg/mL) to the cell monolayer followed by reading using the microplate reader. Anti-T. cruzi activity (IC 50 and IC 90 ) and SI values were determined. All experimental assays were performed at least 3 times in duplicate.

Drug Efficacy in 3D Microtissue
A three-dimensional cardiac model was applied as a cell culture platform to test the efficacy of promising candidates. Cardiac spheroids, with 5-7 days of culture, were infected for 24 h with T. cruzi Dm28c-Luc (5 × 10 5 parasites/well) and then, after washing, incubated for 72 h at 37 • C with 15 to 30 times the IC 90 value of the most active compounds (IC 50 ≤ 10 µM). Microtissues were also incubated with DMSO (0.1%) and Bz (100 µM) as negative and positive controls, respectively. After luciferin addition (300 µg/mL), the luminescence was measured, and the data were expressed as arbitrary luminescence units (ALU).

Washout Assay
A washout assay was performed to evaluate the capacity of the promising candidates to eliminate all parasites. Thus, VERO cells, seeded in 96-well plates at a density of 1.5 × 10 4 cells/well, were infected with T. cruzi (Dm28c-Luc) at a 10:1 parasite-host cell ratio (24 h). Two treatment schedules were performed: 3 days with 30, 50, and 100 µM concentrations and 10 days with 30 and 60 times the IC 50 concentrations. After treatment, the cultures were washed with PBS and kept for the same period in RPMI 1640 medium supplemented with 10% FBS without compound pressure. The culture medium was changed every 3 or 4 days during the time course, and the supernatant was harvested for luminescence reading after luciferin (300 µg/mL) addition. At the endpoint, the monolayers were also evaluated for parasite load. The luminescent signal was read using a Glomax reader. Bz (100 µM) and DMSO (≤1%) were used as positive and negative controls, respectively.

Drug Combination Assay
The combination of promising candidates and Bz was performed using an isobologram method [39]. Initial concentrations were determined by ensuring that IC 50 concentrations of monotherapy compounds remained close to half in serial dilutions (1:3-6 concentrations). Solutions at the initial concentration of each compound were prepared and then mixed in proportions of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5 (v/v) of promising compounds and Bz, respectively. The anti-T. cruzi activity for the serial dilutions (1:3) of the ratios was then analyzed, as previously described [39]. The IC 50 values for each combination were used to determine the fractional inhibitory concentration index (FICI); thus, FICI(A) = IC 50 FICI(B)) and the average of the sums for the FICIs (xΣFICI) were calculated. The FICI values were plotted on an isobologram graph. The xΣFICI was applied as a classification criterion for interactions: as synergistic xΣFICI ≤ 0.5, additive xΣFICI > 0.5-1, without interaction xΣFICI > 1-4, and antagonistic for xΣFICI > 4.

Statistical Analysis
Data are presented as the mean and standard deviation (SD) of at least three independent experiments. All statistical analyses were performed using GraphPad Prism version 8.2 (GraphPad software, Inc., La Jolla, CA, USA). A statistical difference, calculated using an ANOVA (Kruskal-Wallis), was considered as a p-value ≤ 0.05.

In Silico Characterization of 1,2,3-Triazole Derivatives
Computer-aided drug discovery tools have been an attractive strategy for identifying novel hits and optimizing hit-to-lead compounds [40]. In silico analysis has an impact on accelerating the discovery of new drugs by discarding compounds with poor physicochemical and pharmacokinetic properties in the early stages of development, thus reducing the risks of failure [41,42]. Considering this issue, the physicochemical properties of the 1,2,3triazole derivatives were assessed with the aim of predicting the drug-likeness profile of the designed compounds. A total of 14 derivatives (1a-n) were analyzed for their compliance with Lipinski's rule, which considers that a molecule is orally bioavailable when it has a molecular weight (MW) < 500, octanol/water partition coefficient (cLogP) < 5, number of hydrogen bond donors (HBD) < 5, and hydrogen bond acceptors (HBA) < 10. The compounds had a low MW, varying from 205.21 to 300.15 g/mol ( Figure 1). The optimal cLogP range (1.520132.9) was mostly observed in 1,2,3-triazole derivatives, favoring permeation of biological barriers. Only 1i (cLogP = 0.73) and 1n (cLogP = −0.13) had greater polarity (Figure 1), which may favor aqueous solubility but tended to limit membrane permeability. Lipophilicity is an important property that impacts drug absorption, distribution, metabolism, excretion, and toxicity (ADMET) [43]. However, oral bioavailability is also influenced by tPSA and flexibility [44]. Our in silico analysis showed that all derivatives have a topological polar surface area (tPSA) < 140 Å 2 , HBD < 5, HBA < 10, and rotatable bonds (RB) < 10, with a prediction of good oral bioavailability (Figure 1). These findings were also confirmed using bioavailability radar (Supplementary Figure S2), with most of the physicochemical parameters, including lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), flexibility (FLEX), and saturation (INSATU), within the physicochemical space that represents the prediction of oral bioavailability (radar pink area). rotatable bonds (RB) < 10, with a prediction of good oral bioavailability (Figure 1). These findings were also confirmed using bioavailability radar (Supplementary Figure S2), with most of the physicochemical parameters, including lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), flexibility (FLEX), and saturation (INSATU), within the physicochemical space that represents the prediction of oral bioavailability (radar pink area).

Cytotoxicity and Biological Activity
All triazole analogs showed good drug-likeness prediction and advanced to phenotypic screening assays against T. cruzi (Dm28c-Luc). Toxicity is undoubtedly the main cause of failure for drugs in clinical trials. Thus, the cytotoxicity was explored in vitro on VERO cell monolayers, with viability measured using the ATP level. Low toxicity was observed for all compounds analyzed (CC50 > 236 µM) except 1f (CC50 = 86.8 ± 2.73 µM), which demonstrated moderate cytotoxicity (Table 1). Next, we assessed the antiparasitic effect of the 1,2,3-triazole derivatives (1a-n) ( Table 1). Most compounds had low activity against T. cruzi, with IC50 > 70 µM for both trypomastigotes and intracellular amastigotes

Cardiotoxic Effect of 1,2,3-Triazole Candidates
Drug-induced cardiotoxicity remains a major cause of attrition in drug development [52,53]. In this regard, we explored the cardiotoxic effect of promising candidates (1d, 1f, and 1g) in a 2D and 3D primary culture of heart muscle cells. Derivatives 1d and 1g did not induce a cardiotoxic effect, showing CC 50 > 500 µM in both 2D and 3D culture models (Table 2). Derivative 1f revealed a low toxicity (CC 50 = 111.33 ± 10.06 µM) on cardiac monolayers (2D) but no cardiotoxic effect was detected on 3D cardiac microtissue (CC 50 > 500 µM). In general, 2D cultures are more susceptible to compound-induced toxicity than 3D microtissues [54]. The difference in drug response may be related to physical and physiological properties, such as morphology, distribution of surface receptors, proliferative stage, and pH level, promoting drug susceptibility or resistance [54]. Table 2. Cardiotoxic effect of 1,2,3-triazole derivatives.

Drug Efficacy in T. cruzi-Infected 3D Cardiac Spheroid
Bridging the gap between in vitro and animal models in drug discovery, 3D cardiac microtissue, which has a more realistic physiological microenvironment of in vivo tissues compared to 2D cultures [55], was also applied to evaluate the efficacy of the compounds. Organoid cultures have been highlighted as a drug screening platform to improve the efficiency of drug development [56], providing more robust data to proceed with in vivo preclinical studies. Herein, T. cruzi-infected cardiac spheroids were used to address the effectiveness of promising candidates since the cardiac cells are the main target of infection by T. cruzi. The 3D microtissues were treated with the promising candidates at concentrations of 15 to 30 times the IC 50 , except for 1f, whose maximum concentration reached 50 µM. Derivatives 1d and 1f were able to significantly reduce the parasite load in 3D cardiac microtissue, showing an effective diffusion and effectiveness of the 1,2,3-triazole candidates ( Figure 2). These derivatives showed an anti-T. cruzi effect similar to Bz, even at low concentrations (≤50 µM). In contrast, 1g did not effectively decrease the total number of viable intracellular parasites ( Figure 2).  , 1f and 1g) in T. cruzi-infected 3D cardiac spheroids. Anti-T. cruzi activity of the compounds was determined using quantification of the luminescent signal, measured in arbitrary luminescence units (A.L.Us.) Note that 1d and 1f in both concentrations significantly inhibited the viability of the parasites, exhibiting efficacy comparable to Bz. A one-way ANOVA test was used to determine the statistical significance relative to the untreated and treated groups, p ≤ 0.0001 (****).
Our data reinforce using 3D primary cardiac microtissue as a suitable model to assess drug efficacy, which can improve the translation potential for anti-T. cruzi drugs, reducing gaps between in vitro and in vivo models. Efficient preclinical screening is essential to avoid therapeutic failures in clinical development and the use of 3D culture models has been widely encouraged to accurately screen candidate drugs [57]. The 3D culture model has attracted attention in the development of new drugs and vaccines for infectious diseases [58]. This tool has allowed advances in the discovery of antimicrobial [59,60] and  (1d, 1f and 1g) in T. cruzi-infected 3D cardiac spheroids. Anti-T. cruzi activity of the compounds was determined using quantification of the luminescent signal, measured in arbitrary luminescence units (A.L.Us.) Note that 1d and 1f in both concentrations significantly inhibited the viability of the parasites, exhibiting efficacy comparable to Bz. A one-way ANOVA test was used to determine the statistical significance relative to the untreated and treated groups, p ≤ 0.0001 (****).
Our data reinforce using 3D primary cardiac microtissue as a suitable model to assess drug efficacy, which can improve the translation potential for anti-T. cruzi drugs, reducing gaps between in vitro and in vivo models. Efficient preclinical screening is essential to avoid therapeutic failures in clinical development and the use of 3D culture models has been widely encouraged to accurately screen candidate drugs [57]. The 3D culture model has attracted attention in the development of new drugs and vaccines for infectious diseases [58]. This tool has allowed advances in the discovery of antimicrobial [59,60] and antiparasitic drugs, but it has been rarely used in Chagas disease drug screening. H9c2 cardiomyoblast 3D spheroids were recently used to evaluate the anti-T. cruzi effect of nucleoside analogs, showing potent activity against intracellular amastigotes [61]. HepG2 monolayers and 3D cultures were also exploited to investigate the hepatotoxicity of atorvastatin-aminoquinoline derivatives screened against T. cruzi [62]. The efficacy of pyrazole derivatives, pyrazole-imidazoline and pyrazole-thiazoline scaffolds, against T. cruzi was also explored using VERO cell spheroids [38,39]. In cancer research, 3D microtissue has been widely used as a powerful tool for anti-cancer drug screening since its complex organization represents the tumor microenvironment with greater reliability, interfering with drug diffusion and efficacy [63,64]. Therefore, the introduction of 3D culture models in preclinical drug screening platforms may overcome the drawbacks of immortalized cell lineage (2D culture) and have the potential to reduce translational lacunas between in vitro and animal models.

Drug Potential to Inhibit Parasite Resurgence
An important open question was whether treatment with the promising candidates could induce a sterile cure in vitro. To assess drug efficacy more accurately, monolayers of VERO cells infected with T. cruzi (24 h) were exposed to short-term treatment (3 days) followed by cultivation for 3 days without compound pressure. Both the release of trypomastigotes in the culture supernatant and the presence of intracellular amastigotes in cell monolayers were determined after reading the luminescent signal (arbitrary luminescence unit; A.L.U.). Our results demonstrated significant inhibition of released trypomastigotes and cell monolayer infection. Among the promising candidates analyzed, 1d (100 µM) and 1f (50 µM) were the most effective compounds (Figure 3). In contrast, 1f (30 µM) and 1g (100 µM) did not significantly reduce the parasite load. Despite the potent activity of 1d (100 µM) and 1f (50 µM), achieving an approximately 10-to 12-fold reduction, respectively, in parasite load compared to untreated cultures, these derivatives did not prevent parasite resurgence (Figure 3). Treatment with Bz (100 µM) potentially inhibited the infection progression, with a few parasites released and low infection of the cell monolayer (Figure 3), indicating failure to eliminate parasites with short-term treatment.
The promising results showing that the analogs potentially decreased infection without drug pressure led us to investigate their ability to induce sterile cure using a long-term treatment assay. Thus, T. cruzi-infected VERO cell monolayers were treated for 10 days with the promising candidates, and the reversibility was monitored for another 10 days. The maximum concentration of 1d and 1f showed a similar inhibitory effect to Bz on the release of trypomastigotes (Figure 4). Although the 1,2,3-triazole derivatives potentially reduced the parasite load, none inhibited infection reactivation ( Figure 4). However, prolonged treatment with Bz also failed to induce a sterile cure (Figure 4). Treatment of VERO cells infected with Silvio X10/7 with Bz (12.5 to 50 times EC 50 ) for 8 days also did not prevent relapse, but a lack of recrudescence was evidenced for 60 days after 16 days of treatment with 25 to 50 times EC 50 [65]. Interestingly, treatment for 8 days with Bz was able to delay the resurgence of the parasite, even using a highly proliferative strain (Silvio X10/7). This fact was not observed in our analysis, where a very low, but continuous, release of trypomastigotes was evidenced. It is possible that this effect is related to the maximum concentration of Bz used (20 times the IC 50 ) or the susceptibility of T. cruzi.
(100 µM) did not significantly reduce the parasite load. Despite the potent activity of 1d (100 µM) and 1f (50 µM), achieving an approximately 10-to 12-fold reduction, respectively, in parasite load compared to untreated cultures, these derivatives did not prevent parasite resurgence (Figure 3). Treatment with Bz (100 µM) potentially inhibited the infection progression, with a few parasites released and low infection of the cell monolayer (Figure 3), indicating failure to eliminate parasites with short-term treatment. The promising results showing that the analogs potentially decreased infection without drug pressure led us to investigate their ability to induce sterile cure using a longterm treatment assay. Thus, T. cruzi-infected VERO cell monolayers were treated for 10 days with the promising candidates, and the reversibility was monitored for another 10 days. The maximum concentration of 1d and 1f showed a similar inhibitory effect to Bz on the release of trypomastigotes (Figure 4). Although the 1,2,3-triazole derivatives potentially reduced the parasite load, none inhibited infection reactivation ( Figure 4). However, prolonged treatment with Bz also failed to induce a sterile cure (Figure 4). Treatment of VERO cells infected with Silvio X10/7 with Bz (12.5 to 50 times EC50) for 8 days also did not prevent relapse, but a lack of recrudescence was evidenced for 60 days after 16 days of treatment with 25 to 50 times EC50 [65]. Interestingly, treatment for 8 days with Bz was able to delay the resurgence of the parasite, even using a highly proliferative strain (Silvio X10/7). This fact was not observed in our analysis, where a very low, but continuous, Figure 3. Short-term washout assay with 1,2,3-triazole-treated T. cruzi-infected VERO cells. Treatment (3 days) of Dm28c-luc-infected VERO cells with 1d, 1f, and 1g at different concentrations (30, 50, or 100 µM) followed by 3 days in the absence of treatment. Detection of trypomastigotes in the culture supernatant (a) and intracellular parasites in VERO cell monolayers (b) was revealed using arbitrary luminescence units (A.L.Us.). Statistical significance, in relation to the untreated group, was determined using a one-way ANOVA test, with p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**) and p ≤ 0.05 (*).  Drug combinations with distinct pharmacological compounds have been applied as a strategy to improve anti-T. cruzi activity. The resulting in vitro effect of Bz combined with the promising candidates against intracellular amastigote was analyzed with a luminescent assay, using T. cruzi Dm28c-Luc. Drug pairs, combined at six different ratios (0:5, 1:4, 2:3, 3:2, 4:1, and 5:0), allowed the calculation of FICI, ΣFICI, and xΣFIC. The data revealed an additive effect of the 1,2,3-triazole derivatives 1d (xΣFICI = 0.93), 1f (xΣFICI = Figure 4. Long-term (21 days) washout assay with 1,2,3-triazole-treated, T. cruzi-infected VERO cells. Infected monolayers (24 h) were treated for 10 days with promising candidates (1d, 1f, and 1g) followed by another 10 days in the absence of treatment pressure. Detection of trypomastigotes released in the culture supernatant after treatment with 1d (a), 1f (b), and 1g (c) at different concentrations (30 or 60 times IC 50 ). (d) Viable intracellular parasites in the cell monolayer were revealed using arbitrary luminescence units. Statistical significance, in relation to the untreated group, was determined using a one-way ANOVA test, with p ≤ 0.0001 (****) and p ≤ 0.001 (***).

Conclusions
This study integrated virtual analysis and phenotypic drug screening to predict the oral bioavailability and evaluate the trypanocidal effect of 1,2,3-triazole derivatives, respectively. Three-dimensional spheroids and reversibility assay are suitable in vitro preclinical models to improve the translational success of drug candidates. The reported data highlight the activity of 1d, 1f, and 1g against T. cruzi, showing good permeability and efficacy in 3D cardiac microtissue. However, the washout assay demonstrated that while the analogs potentially reduced the parasite load, they did not prevent parasite resurgence. The combination of Bz and triazole promising candidates induced an additive effect with the isobologram analysis, suggesting that combination treatment may lead to a positive outcome in vivo. New 1,2,3-triazole derivatives, containing methoxy and methyl substituents on the phenyl ring, will be designed, aiming to improve the compounds' anti-T. cruzi activity.